Cette étude vise à explorer les mécanismes de tolérance des plantes aux xénobiotiques organiques de la famille des HAPs (phénanthrène), à travers l’analyse de l’impact des évènements de spéciation par hybridation et duplication génomique (allopolyploïdie). Nous avons pour cela mené une approche comparative sur un modèle de spéciation allopolyploïde récente, constitué des espèces parentales hexaploïdes S. alterniflora et S. maritima, et de l’allopolyploïde S. anglica qui résulte de la duplication du génome de leur hybride F1 S. x townsendii. Une approche intégrative basée sur des analyses physiologiques et moléculaires nous a permis de montrer que chez Spartina l’hybridation et le doublement du génome augmentent la tolérance aux xénobiotiques. Le parent paternel S. maritima se montre particulièrement sensible au phénanthrène par rapport au parent maternel S. alterniflora. Différentes analyses transcriptomiques ont permis l’identification de novo de transcrits spécifiquement exprimés en condition de stress, et l’annotation des petits ARNs (miARNs, leurs gènes cibles, et siARNs) agissant en tant que régulateurs de l’expression des gènes et la régulation des éléments transposables. Les analyses d’expression différentielle en réponse au stress ont permis de générer un modèle de régulation (miARN/gènes cibles) en réponse aux HAPs, testé par validation fonctionnelle en système hétérologue chez Arabidopsis. Un travail exploratoire de profilage du microbiome de la rhizosphère des spartines exposées au phénanthrène a été réalisé pour préciser les mécanismes de dégradation des xénobiotiques dans l’environnement en vue d’une application dans les stratégies de remédiation verte. / We explored mechanisms involved in tolerance to organic xenobiotics belonging to PAHs (phenanthrene), in the context of allopolyploid speciation (hybrid genome duplication). We developed a comparative approach, using a recent allopolyploidization model including the hexaploid parental species S. alterniflora and S. maritima, and the allopolyploid S. anglica, which resulted from genome doubling of the F1 hybrid S. x townsendii. Integrative approach based on physiological and molecular analyses highlights that hybridization and genome doubling enhance tolerance to xenobiotics in Spartina. The paternal parent S. maritima exhibits higher sensitivity compared to the maternal parent S. alterniflora. Various transcriptomic analyses were performed, to identify de novo stress responsive transcripts, and to annotate small RNAs (miRNAs, their target genes, and siRNAs) involved in gene expression and transposable element regulations. Differential expression analyses in response to stress allowed us to develop a putative miRNA regulatory network (miRNA/target genes) in response to PAH, functionally validated in Arabidopsis as heterologous system. An exploratory profiling of Spartina rhizosphere microbiome exposed to phenanthrene was also performed to characterize environmental degradation abilities, in the perspective of optimizing green remediation strategies.

Le dogme central de la biologie repose sur la production de protéines à partir de notre ADN. L’ADN est d’abord transcrit en ARN et celui-ci est ensuite traduit en protéine. C’est donc en cette dernière qu’est localisé le “pouvoir exécutif” de la cellule, ce qui explique le fait que les protéines soient devenues le centre d’attention de la recherche. L’ARN, quant à lui, est donc depuis longtemps considéré comme une molécule intermédiaire, dont l’unique raison d’être est le transfert d’information entre l’ADN et les protéines. Pourtant, ces dernières années, les avancées technologiques ont révélé qu’une majeure partie de notre génome, notre ADN, est transcrit en ARNs dits « noncodants » ne donnant pas lieu à une protéine. Ceux-ci sont impliqués dans de nombreux processus cellulaires et de ce fait participent aux pathologies. D’autre part, de nouvelles technologies ont aussi mené à l’observation que le métabolisme des ARNs, codants ou non, est la cible de nouveaux mécanismes de régulation: les modifications chimiques des ribonucléosides. Analysées de manière conjointe, ces découvertes poussent à la révision du rôle des ARNs au sein des processus cellulaires. Dès lors, dans le cadre de cette thèse nous avons voulu mieux comprendre la fonction et la régulation des molécules d’ARN afin d’en révéler le rôle plus central qu’ils jouent dans les processus cellulaire et en particulier, la cancérogenèse. Pour ce faire cette thèse comporte deux parties, la première décrit comment certains ARNs, dit “longs ARNs non-codants” participent au développement et à l’hétérogénéité du cancer colorectal. En effet ces ARNs exercent des fonctions “exécutives” sans être la source d’une protéine. Nous avons identifié 282 long ARNs non-codants dont les profils d’expression reflètent les différentes caractéristiques rencontrées au travers des différents sous-types de tumeurs colorectales. De plus, nos analyses informatiques ont indiqué que ces ARNs font partie intégrante des réseaux de signalisations les plus importants et les plus souvent dérégulés dans les différents sous-types que présente ce cancer. Enfin, et ce via des expériences in vitro nous soutenons la validité de nos analyses informatiques en confirmant le rôle de lncBLID-5, un long ARN non-codant, dans la régulation du cycle cellulaire et de la transition épithéliale vers mésenchymale un processus cellulaire très important dans les cancers colorectaux. Dans la deuxième partie nous avons étudié la méthylation des cytosines de l’ARN, une modification très récemment identifiée. Nous avons découvert que la protéine SRSF2, un facteur général de l’épissage des ARNs, est capable de se lier aux cytosines méthylées et ce plus fortement qu’aux cytosines non-méthylées. Enfin, nous montrons que la mutation P95H de SRSF2, très fréquente chez les patients atteints de leucémie, empêche SRSF2 de favoriser sa liaison aux cytosines méthylées laissant entrevoir de nouvelles explications à l’épissage défectueux conduisant à ce type de cancer. En conclusion nos travaux apportent de nouvelles informations quant à l’implication et la régulation des ARNs codants et non-codants dans le cadre du cancer. Ces résultats devraient nous mener à revoir le rôle qu’occupe l’ARN au sein des processus cellulaires sains ainsi que pathologiques, ouvrant la porte sur une nouvelle dimension de cibles diagnostiques et thérapeutiques. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Les nématodes à galles du genre Meloidogyne sont des parasites obligatoires des plantes capables d’infecter un large panel de plantes d’intérêt agronomique. Ces parasites ont la capacité d’induire la différenciation de cinq à sept cellules racinaires en cellules géantes, hypertrophiées, métaboliquement actives et multinucléées. Ces cellules géantes constituent le site nourricier indispensable au nématode, et sur lequel il va s’alimenter jusqu’à sa reproduction. Le développement de ces cellules entraine une déformation racinaire appelée « galle » qui va perturber l’absorption de nutriments de la plante et l’affaiblir. Des études transcriptomiques ont montré qu’une vaste reprogrammation transcriptionnelle a lieu lors de la formation de la galle. Cette thèse vise à caractériser le rôle des petits ARN, des ARN non codants, au cours de la formation des galles induites par M. incognita. Les petits ARN non codants sont des régulateurs clés de l’expression génique et comprennent deux grandes familles : les microARN (miARN) et les petits ARN interférents (siARN). Pour cela, les petits ARN de racines de la plante modèle Arabidopsis thaliana saines et infectées par le nématode à galle M. incognita ont été caractérisés par séquençage haut débit à 7 et 14 jours après infection, deux stades importants du développement des cellules géantes. Cette étude a permis d’identifier 24 miARN d’Arabidopsis différentiellement exprimés dans les galles en comparaison aux racines non infectées. L’analyse fonctionnelle de ces miARN a permis de valider le profil d’expression dans les galles de cinq miARN et de démontrer le rôle de miR159 dans la réponse de la plante à M. incognita. De plus, une approche pangénomique a été réalisée afin d’identifier les gènes susceptibles d’être régulés par les siARN lors de l’interaction. En conclusion, ce travail a contribué à démontrer d’une part l’implication des miARN dans l’interaction plante - nématode à galles et a permis l’identification des gènes potentiellement régulés par les siARN lors de l’interaction et impliqués dans la formation des cellules géantes induites par les nématodes à galles. / Root-Knot-Nematodes are obligate plant parasites able to infect a large panel of cultivated plants. These parasites have the ability to induce redifferentiation of five to seven root cells into specialized giant cells, hypertrophied, multinucleated and metabolically overactive. These giant cells form the feeding site upon which nematodes feed continuously until reproduction. Giant cells development leads to a root deformation, named gall, which disturbs plant nutrients absorption causing its weakening. Transcriptomic studies showed that a huge transcriptional reprogramming occurs during gall development. This project aims to characterize the role of small RNAs, non-coding RNAs, during gall development induced by M. incognita. Small non-coding RNAs are key regulators of gene expression and include two major families: microRNAs (miRNAs) and small interfering RNAs (siRNAs). Thus, small RNAs from roots of the model plant Arabidopsis thaliana healthy or infected by M. incognita were characterized by Next Generation Sequencing at 7 and 14 days after infection, two important stages of gall development. This study led to the identification of 24 plant microRNAs differentially expressed in galls compared to uninfected roots. Functional analysis of these miRNAs validated the expression pattern in galls of five miRNAs and demonstrated the role of miR159 in the plant response to M. incognita. In addition, a genome-wide approach was used to identify genes that could be regulated by siRNAs during the interaction. In conclusion, this work contributed todemonstrate, on one hand, the involvement of microRNAs in the plant - RKN interaction and allowed the identification of genes potentially regulated by small interfering and involved in the formation of giant cells induced by root-knot nematodes.

No description available.

Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

No description available.

No description available.

Almost every precursor mRNA (pre-mRNA) in a eukaryotic organism undergoes splicing, in some cases resulting in the formation of more than one splice variant, a process called alternative splicing. RNA-Seq provides a major opportunity to capture the state of the transcriptome, which includes the detection of alternative spicing events. Alternative splicing is a highly regulated process occurring in a complex machinery called the spliceosome. In this dissertation, I focus on identification of different splice variants and splicing factors that are produced during Arabidopsis and soybean embryo development. I developed several data analysis pipelines for the detection and the functional characterization of active splice variants and splicing factors that arise during embryo development. The main goal of this dissertation was to identify transcriptional changes associated with specific stages of embryo development and infer possible associations between known regulatory genes and their targets. We identified several instances of exon skipping and intron retention as products of alternative splicing. The coding potential of the splice variants were evaluated using CodeWise. I developed CodeWise, a weighted support vector machine classifier to assess the coding potential of novel transcripts with respect to RNA secondary structure free energy, conserved domains, and sequence properties. We also examined the effect of alternative splicing on the domain composition of resulting protein isoforms. The majority of splice variants pairs encode proteins with identical domains or similar domains with truncation and in less than 10% of the cases alternative splicing results in gain or loss of a conserved domain. I constructed several possible regulatory networks that occur at specific stages of embryo development. In addition, in order to gain a better understanding of splicing regulation, we developed the concept of co-splicing networks, as a group of transcripts containing common RNA-binding motifs, which are co-expressed with a specific splicing factor. For this purpose, I developed a multi-stage analysis pipeline to integrate the co-expression networks with de novo RNA binding motif discovery at inferred splice sites, resulting in the identification of specific splicing factors and the corresponding cis-regulatory sequences that cause the production of splice variants. This approach resulted in the development of several novel hypotheses about the regulation of minor and major splicing in developing Arabidopsis embryos. In summary, this dissertation provides a comprehensive view of splicing regulation in Arabidopsis and soybean embryo development using computational analysis. / Ph. D.

No / The hair follicle is a cyclic biological system that progresses through stages of growth, regression, and quiescence, which involves dynamic changes in a program of gene regulation. Micro-RNAs (miRNAs) are critically important for the control of gene expression and silencing. Here, we show that global miRNA expression in the skin markedly changes during distinct stages of the hair cycle in mice. Furthermore, we show that expression of miR-31 markedly increases during anagen and decreases during catagen and telogen. Administration of antisense miR-31 inhibitor into mouse skin during the early- and midanagen phases of the hair cycle results in accelerated anagen development, and altered differentiation of hair matrix keratinocytes and hair shaft formation. Microarray, qRT-PCR and Western blot analyses revealed that miR-31 negatively regulates expression of Fgf10, the components of Wnt and BMP signaling pathways Sclerostin and BAMBI, and Dlx3 transcription factor, as well as selected keratin genes, both in vitro and in vivo. Using luciferase reporter assay, we show that Krt16, Krt17, Dlx3, and Fgf10 serve as direct miR-31 targets. Thus, by targeting a number of growth regulatory molecules and cytoskeletal proteins, miR-31 is involved in establishing an optimal balance of gene expression in the hair follicle required for its proper growth and hair fiber formation.

Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer. / Ph. D. / Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using high throughput genetic screens identified genes that regulate the survival of cancer cells from a deadly type of brain cancer called glioblastoma (GBM). Another screen revealed genes that regulate a process called autophagy in cancer cells from a type of leukemia called chronic myelogenous leukemia (CML). Autophagy is a process that cancer cells can use to survive through chemotherapy. One screen identified the gene Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit β (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, normal and cancerous, where cellular components are broken down and reassimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in different types of cancer. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.